Received May 27, 2014
According to a modern look acid-sensing ion channels (ASICs) are one of
the most important receptors that perceive pH change in the body. ASICs
represent proton-gated Na+-selective channels, which are
expressed in neurons of the central and peripheral nervous system.
These channels are attracting attention of researchers around the
world, as they are involved in various physiological processes in the
body. Drop of pH may occur in tissues in norm (e.g. the accumulation of
lactic acid, the release of protons upon ATP hydrolysis) and pathology
(inflammation, ischemic stroke, tissue damage and seizure). These
processes are accompanied by unpleasant pain sensations, which may be
short-lived or can lead to chronic inflammatory diseases. Modulators of
ASIC channels activity are potential candidates for new effective
analgesic and neuroprotection drugs. This review summarizes available
information about structure, function, and physiological role of ASIC
channels. In addition a description of all known ligands of these
channels and their practical relevance is provided.
KEY WORDS: acid-sensing ion channel, pain perception, ligand, low
molecular weight modulator, peptide

One of the most urgent tasks in biochemistry is to search for and/or to
create molecular tools to study the mechanisms of functioning of the
sensory systems of living organisms both in normal and pathological
states. Interesting objects for study are the acid-sensing ion channels
(ASIC) related to the superfamily of amiloride-sensitive
degenerin/epithelial Na+-channels (DEG/ENaC) [1].

ASIC channels are found in large numbers in neurons of the central
nervous system (CNS) [2, 3],
where at least three (ASIC1a, ASIC2a, and ASIC2b) subunits from the six
known are found. Among all the subunits present in brain, ASIC1a is
basic. Homomeric ASIC1a and heteromeric ASIC1a/2b channels are able to
conduct both Na+ and Ca2+ [4,
5]. It was shown that ASIC1a and ASIC2 are directly
involved in synaptic plasticity, learning, transmission of nerve
excitation [3, 6], ischemia,
and neuronal cell death [7-10], as well as epilepsy [11].

In neurons of the peripheral nervous system, homomeric ASIC3- and
heteromeric ASIC3-containing channels are mainly present [12]. These channels are shown to: a) participate in
perception of acid-mediated, inflammatory, or postoperative pain [13-15]; b) contribute to
development of primary and/or secondary mechanical hypersensitivity in
muscles [16]; c) participate in cutaneous and
visceral mechanical sensitivity and in perception of pain from
mechanical stimuli [17-19]; d) be involved in perception of pain
signals from the lungs and gastrointestinal tract [20].

STRUCTURE OF ASICs

Neuronal receptors able to perceive extracellular pH decrease were
discovered in 1980 [21], but they were cloned and
characterized only in 1997 [22]. In mammals, the
presence of four genes (ACCN 1-4) encoding at least six subunits
of these channels (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4)
was established [23]. The primary structure of
ASIC channels is quite conservative for a variety of mammalian species
(rats, mice, and humans). Thus, ASIC isoforms in rats have amino acid
sequence similarity of 45-80%. In addition to mammals, ASICs have been
found in animals of other classes such as toadfish (Batrachoididae),
lampreys, sharks, and the freshwater aquarium fish Danio rerio
[24, 25].

As in all DEG/ENaC channels, membrane topology of the individual subunit
of the ASIC channel consists of two transmembrane domains (TM1 and TM2)
linked via a large cysteine-rich extracellular loop with short
intracellular N- and C-terminal regions [30]. The domain of the N-terminal region,
adjacent to TM1, is responsible for channel selectivity to
Na+ [31]. The conducting pore in the
membrane is formed by direct contacts of the TM1 and TM2 domains of all
three subunits [32]. The TM2 domain is highly
conserved not only among the ASIC channels, but also among channels of
the DEG/ENaC family in general [33]. It is
involved in the formation of the desensitization gate. Amino acid
residues (a.a.) Asp433-Gly436 are important for it, and the Asp433 side
group is directed into the lumen of the pore [27].
TM2 also contains an amiloride-binding site and a selective filter
formed by a.a. 443-445 [32, 33], as well as two amino acid residues responsible
for blocking by Ca2+ (channel ASIC1a) [34].

The extracellular region of each subunit has the shape of a “hand
clutching a ball” consisting of “wrist”,
“palm”, “finger”, “knuckle”, and
“β-ball” domains and a cystine-rich
“thumb” domain (Fig. 1) [26, 27]. It is formed by seven
α-helices (α1-α7) and 12 β-layers
(β1-β12) [26] and stabilized by 14
disulfide bonds, which are quite conserved among DEG/ENaC channels [35]. The palm domain contains seven β-strands,
it is a central element of the extracellular region and forms numerous
contacts with other domains. Part of the extracellular loop near TM1
contains the degenerin site [36]. The
extracellular part is connected with the TM domains through the small
wrist domain consisting of only two ordered loops.

Numerous studies have established an important functional role for the
various amino acid residues. The β10-layer in palm domain and a
region located between the α6- and α7-helices in the
knuckle domain contain two conserved glycosylation sites Asn367 and
Asn394 that play an important structural role in proton-sensing [26, 30, 37]. Residues Asp107 in the α1- and Arg153 in
the α3-helices of the finger domain in the ASIC3 channel form an
acid–base interaction [38]. The Trp287
residue of the thumb domain and Tyr71 of the wrist domain in ASIC1
interact with each other and participate in channel opening [39]. The Asp79-Glu80 pair is conserved in all ASIC
channel subunits, it is associated with inactivation of the ASIC3
channel [40] and in addition influences
proton-sensitivity in the ASIC2a channel [41].
His72 is important for the sensing pH changes [42]
and is in close proximity to the domain responsible for the
desensitization kinetics [43].

At the interface between the thumb, finger, and palm domains, chloride
ions are accessed to each subunit while being coordinated by Arg310,
Glu314, and Lys212 residues of the neighboring subunit [26, 27]. As shown by experiments
with the replacement of chlorine ions and mutagenesis of coordinating
amino acid residues, access of Cl– to the channel
plays an important role in the channel desensitization [44].

A proton-sensitive sensor is located at the interface between the finger
and thumb domains of one subunit and the palm domain of the neighboring
subunit. This area, which looks like a cavity on the surface of the
channel, is rich in acidic residues, so it was named the “acidic
pocket” [26, 41, 45]. Cavities have strongly expressed negative
electrostatic potentials due to the presence of 12 acidic residues
– Asp79, Glu420, Glu426, Asp433 in the upper part and Glu80,
Glu374, Glu412, Glu417 in the central part of the pore. It is supposed
that the cavities serve as “cationic reservoirs”
concentrating cations near the pore of the ion channel, which provides
reliable channel conductivity [46]. Engaging
bivalent cations to the cavity reduces the concentration of
Na+ near the pore and thus prevents Na+ current.
This is observed for ASIC3 at the presence of Ca2+ [47].

It has also been found that the loop region connecting the β9-layer
of the palm domain and the α4-helix of the thumb domain plays an
important role in signal transduction from the extracellular region to
the transmembrane domains, resulting in a conformational rearrangement
and opening of the channel [48]. Proline residues
located in this loop form a sharp turn followed by the approach of
Trp287 and Tyr71 toward each other in vicinity of TM1 [49]. Conformational changes occur also in the wrist
domain, which presumably lead to the subsequent twisting (or bending)
of TM1 and TM2. During the transition of the channel from the closed to
the open or desensitized state, important conformational changes occur
also in other domains. Thus, mutagenesis of various residues of human
ASIC1a (hASIC1a) identified a number of residues that alter the
kinetics of the opening and closing of the pore: in the region between
the β1-β2-layers and the β1-layer of the palm domain; in
the loop between the palm and finger domains; in the loop between the
β-ball and finger domains; and in the finger domain [40, 43, 50-52].

Based on generalization of the results of numerous experiments, the
following general mechanism of opening of the channel in response to a
change in pH was suggested: protonation/deprotonation of acidic amino
acid residues of the thumb domain affects its interaction with amino
acid residues located in the finger domain, which moves the thumb
domain. Then the signal through the wrist domain is transmitted to the
TM domains, which leads to a change in the inclination angle of the
helices, expansion of the extracellular part of the pore, narrowing in
the selective filter, and channel opening [32].
Pore opening leads to the fact that the backbone carbonyl oxygen atoms
as well as the side chain oxygen atoms of residues such as Asp433 align
along the third order symmetry axis on the path of sodium ions. Long
duration of low pH causes the mutual convergence of the wrist domain
and the neighboring β1 and β12 strands, which leads to
occlusion of the central region of the pore and closure of the ion
channel (the desensitization).

ELECTROPHYSIOLOGICAL PROPERTIES OF ASICs

The electrophysiological properties and pharmacological profiles of the
ASIC channels have been carefully studied in heterologous expression
systems [53] and in neurons of different areas of
the brain such as cerebral cortex [9], hippocampus
[29], striatum [54],
cerebellum [55], net ganglia [56], and spinal cord [57].
Figure 2 shows typical examples of ASIC currents
corresponding to homomeric ASIC1a, 1b, 2a, and 3 channels expressed in
oocytes of the clawed frog Xenopus laevis. The remaining
subunits do not form homomeric actively conducting sodium channels.
Homomeric ASIC3 channels are fundamentally different in the form of the
response to stimulating pulse. They have a two-component response to pH
drop in the extracellular medium. These responses have the form of a
fast-inactivating current with greater amplitude (transient component
or peak) and slow-inactivating current with a smaller amplitude
(sustained component or plateau).

Fig. 2. Characteristic incoming currents of ASIC channels.
Measurements were carried out on oocytes of the frog Xenopus
laevis expressing homomeric channels of rat in the configuration of
the whole cell. Activation of the ion current was caused by a rapid
replacement of solutions with different values of pH (data obtained in
the Laboratory of Neuroreceptors and Neuroregulators, Institute of
Bioorganic Chemistry, Russian Academy of Sciences).

The amino acid sequence similarity between the subunits ASIC1a and
ASIC1b is approximately 70%, but ASIC1b with a similar form of the
recorded current differs in many other parameters. First, ASIC1b
channels are found in mammals only in peripheral sensory neurons [58]. In the example found in rodents, they are
impermeable to Ca2+, while ASIC1a channels conduct these
ions [4]. It is functionally important that ASIC1b
channels are activated at a low pH (~6.5 vs. ~7.0 for ASIC1a) and,
accordingly, have lower pH50 (pH at which the activation of
the channel reaches half of the maximum value) values (~5.9 vs. 6.8 for
ASIC1a) [22].

Homomeric ASIC2a channels are relatively insensitive to protons (their
pH50 value is 4.4) [59]. Functional
heteromeric channels ASIC2a/1a are found in mammalian brain [9, 29, 54].
Unlike ASIC2a subunits, homomeric ASIC2b subunits do not form
functional channels in vivo; they are always present in a complex with
other ASIC subunits [5, 53, 59]. For example, channels ASIC2b/1a are involved in
triggering neuronal cell death in acidosis caused by ischemia [5].

ASIC3, like ASIC1b, is found mainly in peripheral sensory neurons [57, 60-62].
Moreover, it was shown that ASIC3 from sensory neurons of rat skin
gives predominant response under moderate acidification [13]. Electrophysiological data showed that in sensory
neurons ASIC3 subunits function as homomeric and heteromeric channels
[28, 62, 63]. They are susceptible to acidification both in
physiological and pathological processes, such as skin sensitivity,
pain perception under inflammatory processes, and ischemia [60, 64-68].

As noted earlier, these channels have a unique two-component response to
acidic stimulation. The transient component of the current is highly
sensitive to protons (pH50 6.5), has a large amplitude, and
is rapidly desensitized [53, 61]. If there is a drop in pH value from 7.4 to 6.5
and <6.0, then there is a slowly developing component of the current
ASIC3, which lasts as long as the acidic stimulation [8, 22, 69].
In the generation of the plateau effect for different intervals of pH
(i.e. >6.5 and <6.0), various structural elements of the channel are
involved [69]. Thus, depending on the intensity
and duration of the acid stimulus, the form of the recorded current
through ASIC3 can be different: transient component, sustained
component, or the sum of both components.

ASIC4 subunits are present in the hypophysis. Like ASIC2b, they do not
form functional homomeric channels [70].

ASICs are characterized by the fact that they can go into a state of
steady-state desensitization (also known as steady-state inactivation).
This process occurs under a weak but long-lasting increasing proton
concentration for a period from a few seconds to a few minutes [47, 71]. Thus, subsequent
activation by low pH does not result in a significant current. As in
the case of the activation, stationary desensitization is mediated by
addition of a proton to specific sites in the extracellular loop. These
inactivation sites have a higher degree of affinity for protons than
sites that are responsible for activation of the channel [71].

ROLE OF ASICs IN PHYSIOLOGICAL PROCESSES

ASIC channels have been found in the postsynaptic membrane and bodies of
CNS neurons [6]. Interactions of these channels
with the synapse-associated protein PSD-95 (which plays an important
role in synaptic plasticity) and kinase 1-interacting proteins have
been demonstrated [72, 73].
These facts and the fact that the pH of the synaptic cleft is changed
during operation of the synapse suggest that the ASICs can affect
synaptic transmission. This was demonstrated by the example in mice of
hippocampal neuron knockout of the ASIC1a channel, which showed the
absence of long-term potentiation of excitatory postsynaptic potential
(EPSP) produced by high-frequency stimulation, in contrast to wild-type
mice [6]. With the same mouse knockout model, the
absence of inhibitory effect of NMDA-receptor antagonists,
D-2-amino-5-phosphonovalerate, on EPSP summation was shown, unlike
wild-type mice, which demonstrated a synergistic effect in functioning
of ASIC1a channels and NMDA-receptors [74].

ASIC1a channels were found in the amygdala, which plays an important
role in anxiety behavior [3]. It was shown that
mice with knockout of the gene encoding ASIC1a were considerably less
susceptible to anxiety in the “open field” test in response
to sudden noise or predator odor [75]. In this
case, increased expression of the channels leads, on the contrary, to
increased anxiety [76], but it had no effect on
the appearance of undue anxiety [77].

The role of ASIC1a channels in the development of
CO2-mediated anxious behavior was also established. It was
shown that an increase in CO2 in the air breathed by
experimental mice resulted in a decrease in pH in the brain and caused
the development of anxious behavior. Adding a buffer solution
maintaining a normal pH in the amygdala region significantly attenuated
the symptoms of anxious behavior, but pH decrease in this region
reproduced the effect of CO2. It has been demonstrated that
the elimination/inhibition of ASIC1a channel leads to a substantial
reduction in alarm activity, whereas overexpression of ASIC1a channel
in the amygdala of mice deficient for these channels, conversely,
restores CO2-mediated anxious behavior. Thus, a possible
molecular mechanism underlying the development of fear and anxiety was
shown [78].

The ability of ASICs to sense the change in environmental pH and to
generate an ion current across the cell membrane includes this type of
receptor among the major sensory receptors. The dominant role in the
perception of external stimuli in mammals belongs to ASIC3. In rodents,
ASIC3 channels are present in more than half of muscle afferents and in
more than a third of DRG neurons innervating the knee joint [66, 68]. They were found in
specialized cutaneous mechanosensory structures such as
Meissner’s corpuscle, Merkel nerve endings, free nerve endings,
and lanceolate nerve endings surrounding the hair follicle [65]. The role of ASIC3 in mechanosensing of the
gastrointestinal tract has been described [17, 18].

Mice with a targeted ACCN3 deletion (the gene encoding ASIC3)
– asic3–/– mice – showed increased
sensitivity to weak mechanical stimuli [65, 79]. It was also shown that stomatin-like protein 3
(SLP3), which is involved in response to the touch in mice, in
vitro is associated with ASIC3 and does not interact with the TRPV1
receptor that also plays an important sensory role. SLP3 inhibited
ASIC-mediated currents in sensory neurons that indicates participation
of ASIC3 in mechanosensory complexes [80]. There
is still an absence of evidence about direct activation of ASIC3
channels by mechanical stimuli, leaving open the question of their
direct involvement in mechanosensing [81].

ASIC3 channels have been shown to participate in the processes of
auditory perception and vision. ASIC3 channels have been found in cells
of the spiral ganglion and the organ of Corti, located in the cochlea.
ASIC3-deficient mice showed hearing loss in the early stages of
development, with its subsequent restoration at the age of two months
[82]. ASIC3 inactivation led to negative effects
similar to those in glaucoma and chronic ischemia associated with
defects in the inner segment of “rods” [83]. This is because the signal transmission in
neurons of the retina is strongly dependent on the pH of the
extracellular medium [84].

Besides, ASIC3 channels certainly play an important role in
chemoperception, sensing signals in the form of reduction of the
extracellular medium pH, of Ca2+ level, and of certain
metabolites. For example, asic3–/– mice were
better protected against age-dependent glucose intolerance and
demonstrated an increased sensitivity to insulin. Similar results were
obtained on administration in wild-type mice an inhibitor of ASIC3,
peptide APETx2, which proved the participation of ASIC3 in the
development of age-dependent glucose intolerance and insulin resistance
[85].

Peripheral ASIC3 channels play an important role in the perception of
pain and signal integration under the inflammation process. Moderate
acidosis in combination with hyperosmolarity and arachidonic acid
induces inflammation-associated pain, which declines under the action
of the peptide APETx2 and ASIC3-specific antisense RNA [13]. Also, ASIC3-deficient mice showed almost no
development of thermal and mechanical hypersensitivity during
inflammation caused by injection of complete Freund’s adjuvant or
carrageenan into footpad [15, 86].

ASICs AND PATHOLOGICAL STATES

Many experiments with models of neurodegenerative diseases affecting the
CNS organs have shown that ASICs make a significant contribution to the
perception of damaging stimuli. Thus, in experimental models of
autoimmune encephalomyelitis in mice not expressing ASIC1a, a reduction
in the level of axonal degeneration in comparison with wild-type mice
was shown. Measurements of cerebrospinal fluid pH in mice suffering
from autoimmune encephalomyelitis showed acidification sufficient to
activate the ASIC1a channel, and deletion of the gene encoding ASIC1a
demonstrated a protective effect on neuronal explants in vitro.
Blockade of ASIC channels by amiloride induced the same neuroprotective
effect [10]. Analogously, it has been demonstrated
that the blockade of ASIC1 by amiloride protects both myelin and
neurons against damage in models of acute pain [87]. Thus, a role of ASIC1a in axonal degeneration
was demonstrated, which allowed to propose the use of ASIC1a inhibitors
for neuro- and myelo-protection in multiple sclerosis [87].

Parkinson’s disease is characterized by impaired motor function
and loss of dopaminergic neurons in the substantia nigra [88]. Although the mechanism of neuronal damage is not
clear, there is evidence that the affected neurons express ASIC1a
channels [3, 89]. Blockade of
this channel type by amiloride and psalmotoxin 1 (PcTx1) provided
neuroprotection of the substantia nigra in a mouse model of
Parkinson’s disease induced by
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [90].

Anxiety disorders are debilitating neuropsychiatric diseases. They are
treated mainly with benzodiazepines and selective serotonin reuptake
inhibitors, but these drugs have a variety of side effects. The use of
ASIC1a inhibitors such as PcTx1, A-317567, and amiloride improved
parameters associated with the vegetative nervous system and behavioral
parameters in models of anxiety in rodents [91].

Under intensive neuronal excitation in epileptic seizures, acidification
in the brain was observed [92, 93]. This suggests the participation of ASIC1
channels in epilepsy. In models of epileptic seizures, it was shown
that ASIC1a inhibitors significantly reduce the strength and frequency
of action potentials and prolonged membrane depolarization [94, 95]. However, in other
studies, i.e. on GABA-ergic interneurons, an inhibitory effect exerted
by ASIC1a on the development of seizure was demonstrated [11]. These results suggest that the role of ASIC
channels, in particular ASIC1a, in epileptic seizures is complicated
and ambiguous, depending on numerous factors such as the nature of the
stimulus inducing a seizure, localization in the brain, as well as the
age of neurons [96].

Accumulation of lactic acid during anaerobic hydrolysis as well as the
release of protons from ATP hydrolysis leads to a pH decrease and
acidosis of the brain [97] that plays an important
role in brain injury during ischemia [98] and is
directly related to the strength of myocardial infarction [99]. In cultures of mouse and human cortical neurons,
for example, activation of ASIC channels by a solution of low pH led to
neuronal damage that was inhibited by ASIC1a-specific blockade and by
deletion of the ASIC1a-encoding gene [100]. In
models of cerebral ischemia in rodents, intracerebroventricular
administration of an ASIC1a blocker reduced the strength of stroke by
more than 60% [9, 101]. A
similar neuroprotective effect was observed in mice with knockout of
the ASIC1a-encoding gene [9].

Similarly, ASIC3 channels in the peripheral nervous system are strongly
associated with pathologies of various organs. Studies have shown that
decreasing pH to 6.7 activates ASIC3 channels in neurons innervating
the cardiac muscle. Based on indirect evidence (effects of low pH and
lactic acid), an important sensory role of ASIC3 channels in cardiac
ischemia and angina was proposed [8].
Representatives of ASIC channels, particularly ASIC3, are considered as
important participants in the perception of visceral pain from
mechanical stimuli in the organs of the gastrointestinal tract [18]. In several models of acute and chronic pain
(such as thermal, chemical, mechanical, inflammatory, and neuropathy)
in rodents, an important contribution of ASIC1a to the development of
pain has been demonstrated. Intrathecal or intracerebroventricular
administration of a specific ASIC1a inhibitor, PcTx1, or introduction
of antisense oligonucleotides leads to a significant analgesic effect
[102, 103]. It was found
that inhibition of ASIC1a activates an endogenous enkephalin pathway
that increases levels of Met-enkephalin in the cerebrospinal fluid,
apparently leading to the analgesic effect [102].

MODULATORS OF ASICs ACTIVITY

As indicated in the previous two sections, ASICs, especially types 1a
and 3, are involved in a wide range of processes in animals. Their
malfunction leads to a variety of pathological states that can be
corrected and studied in detail through different ligands. A quite a
large number of endogenous and exogenous ligands are known that can
directly activate acid-sensitive channels or potentiate/inhibit
proton-induced currents through the channels. In the table, we
summarized all published ASIC modulators. These molecules have
different chemical nature and not always selectively interact with
ASICs. Low molecular weight compounds (their structures are shown in
Fig. 3) are now of the greatest practical
interest.

Pharmacology of homomeric ASIC channels*
Note: ↓ inhibitory effect; ↑ potentiating
effect; ☼ activation.
* Results presented in the table were obtained in different experimental
systems with various pH-activating stimuli (see original works for
conditions).

Fig. 3. Low molecular weight modulators of
ASICs.

Endogenous ligands.Arachidonic acid (AA) is a
polyunsaturated fatty acid present in phospholipids of all cell
membranes. It functions not only as a secondary messenger, but it also
plays an important role in various pathological conditions such as
inflammation and ischemic brain injury [131]. It
was found that AA enhances activation of ASIC channels. Studies on
Purkinje cells showed that preincubation with 5-10 µM AA
solution increased the amplitude of both the transient and sustained
components of the ASIC current [55].

Serotonin (or 5-hydroxytryptamine), a proinflammatory agent, has
a potentiating effect on the sustained component of the ASIC3 current.
This effect is observed on activation at pH 6.0 and below. In a model
of inflammatory pain in mice, serotonin showed a significant
enhancement of the pain effect that was not observed in mice deficient
in the gene encoding ASIC3 [119].

Tetrapeptide FMRF-amide and similar peptides are capable of
potentiating H+-mediated currents (EC50 ~
10-50 µM for FMRF-amide) [121].
FMRF-amide does not occur in mammals, but other peptides of this group
such as NPFF, NPAF, and NPSF are present in many parts of the CNS. In
chronic inflammation, elevated level of NPFF is observed in the spinal
cord. NPFF is also found in small and medium-sized DRG neurons. It is
assumed that the modulation of ASICs by endogenous RF-amide peptides is
a response to a strong acidification of the environment. RF-amides
interact with the extracellular domain, which results in an increase in
the current amplitude and deceleration of desensitization of ASIC1 and
ASIC3 (but not ASIC2a) due to decrease of proton sensitivity for sites
responsible for steady-state desensitization [121, 122, 132, 133].

Dynorphins are a class of endogenous opioid neuropeptides
abundantly represented in the CNS. They are involved in various
physiological processes including analgesia and neuroendocrine signal
transmission. At high concentrations, dynorphin A potentiates
acidic pH-activated currents in cortical neurons as well as in CHO
cells expressing homomeric ASIC1a [123]. In the
case of expressed channels, it was shown that the potentiation effect
is mediated by a limiting action on the steady-state desensitization
process and does not depend on the activity of the opioid receptors,
i.e. it occurs under direct action of the peptides on the ASIC1a
channel. At the same time, the depressing effect that dynorphin exerted
on the steady-state desensitization negatively influenced neurons
exposed to prolonged acidosis [123]. It is
therefore considered that dynorphin might exacerbate brain damage in
pathological conditions associated with ischemia.

ASIC channels are modulated by divalent cations, especially
Ca2+ ions, which play an important role in the
regulation of various voltage-dependent and ligand-gated ion channels.
The effect of Ca2+ concentration changes in the
extracellular medium on ASICs depends on whether Ca2+ was
present on channel activation or not. Application of Ca2+
together with acid solution inhibits activation of the channels [25, 61]. Conversely, the absence
of Ca2+ enhances activation of ASICs by an acid solution [71, 134]. Studies on the
mechanisms underlying the modulating action of Ca2+ revealed
that Ca2+ reduces the affinity of the channels (for example,
ASIC3) to H+ ions [71]. It was
suggested that at pH 7.4 ASIC3 channels are closed due to the
blockade by Ca2+. By acidification protons attach to the
channel, which causes dissociation of Ca2+ from its binding
sites and as a consequence an opening of the channel [47]. ASIC1a was found to have two
Ca2+-binding sites: one serves for pore blockade by
Ca2+ and the other mediates Ca-dependent regulation of
activation by protons [34, 135].

At physiological pH and Ca2+ concentration, zinc ions
also play an important role in the regulation of ASIC channels. In CHO
cells expressing different combinations of ASIC subunits, it was shown
that Zn2+ in nanomolar concentration inhibits currents
generated by homomeric ASIC1a and heteromeric ASIC1a/2a channels. This
cation led to both a decrease in the amplitude of the current and to
reduced affinity of ASIC1a to protons. It was shown that Lys133,
located at the beginning of the α2-helix in the finger domain, is
involved in a high-affinity process of inhibition by Zn2+
[105]. At micromolar concentrations,
Zn2+ caused a dose-dependent inhibition of ASIC1b
(IC50 26 µM) [106] and
ASIC3 channels (IC50 61 µM) [107]. The inhibitory effect was also due to the
interaction of Zn2+ with the binding site located in the
extracellular portion of ASIC1b and ASIC3 channels. Experiments with
mutagenesis of the channels proved that Cys149 residue located in the
finger domain of ASIC1b subunit determines the sensitivity to
Zn2+ [106]. At concentrations greater
than 100 µM, Zn2+ interacts also with the
low-affinity binding site on the ASIC2a subunit, which increases
affinity for protons and potentiates activity of ASIC2a-containing
channels [42].

Copper ions (Cu2+) have a modulating effect on ASIC
channels, as shown in cell cultures of hypothalamus, hippocampus, and
cortical neurons. Copper dose-dependently reduces the amplitude of ASIC
currents as well as slowing desensitization [108]. Micromolar concentrations of copper ions
weaken acid-induced membrane depolarization, so Cu2+ can be
regarded as an endogenous modulator that reduces increased neuronal
excitability [108].

Spermine (a polyvalent cation) exerts a potentiating
effect on the activity of ASIC1b and ASIC1a channels [71]. Spermine exacerbates damage of neurons during
ischemia through increasing ASIC1a sensitivity to extracellular medium
acidification [136]. Pharmacological blockade of
ASIC1a or deletion of the gene encoding ASIC1 greatly reduces the
effect of spermine on neuron damage during ischemia in a culture of
dissociated neurons and in a model of focal ischemia in mice [136]. Spermine reduces the desensitization of ASIC1a
in the open state, but also accelerates the recovery after
desensitization in response to repeated acid stimuli. Functionally,
increase in channel activity is accompanied by increased acid-induced
depolarization of neurons and overloading of the cytoplasm by
Ca2+, which may explain the increased negative effect
spermine on neuron damage. Therefore, spermine significantly
contributes to neuron damage in ischemia, in part by enhancing the
activity of ASIC1a receptors.

Exogenous ligands.Amiloride – a nonspecific blocker
of Na+ channels – is used in medicine as a
K+-sparing diuretic agent, but it is also widely used as an
inhibitor of ASICs in research. According to data from many studies, it
inhibits the ion current conducted by these channels, with
IC50 values of 10-60 µM. In the case of ASIC3,
amiloride induces a complex response. Only the peak current component
is inhibited, whereas for the plateau component no effect at low
concentrations and potentiation at higher concentrations
(EC50 of 0.5 mM and pH 7.4-6.8) were observed [8, 9, 22, 54, 58, 112]. Based on the data obtained in the study of
ENaC, it was proposed that the mechanism of the inhibitory effect of
amiloride is a direct blocking the cation-conducting pore of the
channel [137]. A site located in front of the TM2
domain plays an important role in the action of amiloride. Mutation of
residue Gly430 in this area greatly changes the sensitivity of ASIC2a
to amiloride [36]. Amiloride inhibits acid-induced
pain in the peripheral sensory system [86, 138] and acidosis-mediated neuronal damage in the
CNS [4, 9]. However, because of
its adverse effect on ion channels such as ENaC and T-type
Ca2+ channels as well as its action on ion-exchange systems
(Na+/H+- and
Na+/Ca2+-exchangers) it seems unlikely that
amiloride can be used as a neuroprotective agent in the future.

A-317567 – a small molecule vaguely reminiscent of the
amiloride structure is another nonselective blocker of ASICs. In DRG
neurons of rats, this molecule inhibits ASIC1a, ASIC2a, and ASIC3 with
IC50 value of 2-30 µM [113]. In the case of ASIC3, it was shown that
A-317567 blocks both the peak and sustained current components. Side
effects of A-317567 versus amiloride are minimal; it does not show any
diuretic or natriuretic activity, suggests its higher specificity to
the ASIC. A-317567 can be considered as a perspective analgesic because
it has been shown effective for inhibiting pain in rats in a model of
thermal hypersensitivity at a dose of 5-10 mg/kg of the
animal’s weight, which is a 10-fold lower dose than that of
amiloride [113].

Another cation, Gd3+(gadolinium), exerts an
inhibitory effect on both the peak and the sustained component of the
current of homomeric ASIC3 channels and is able to inhibit heteromeric
ASIC2a/3 [109]. It is known that gadolinium ions
block activation of neurons in response to stretching [139]. On this basis, it has been suggested that
inhibition of ASIC3 and ASIC2a/3 currents by Gd3+ may be
evidence of the participation of ASIC3-containing channels in the
perception of mechanical stimuli [109].

Nonsteroidal antiinflammatory drugs (NSAIDs) are widely
used as antiinflammatory and analgesic agents. They inhibit the
synthesis of prostaglandins, which play a major role in inflammatory
processes in tissues. It was shown that NSAIDs inhibit the activity of
ASIC channels at concentrations relevant to their analgesic doses.
Ibuprofen and flurbiprofen, for example, inhibit ASIC1a-containing
channels with IC50 value of 350 µM (measured on
COS line cells). Aspirin and salicylate inhibit ASIC3-containing
channels with IC50 value of 260 µM (measured on
culture of DRG neurons), while diclofenac inhibits ASIC3-containing
channels with IC50 value of 92 µM (measured on
COS line cells) [114]. In this case, aspirin,
salicylate, and diclofenac suppress only the sustained component of the
ASIC3 current. It is also known that NSAIDs inhibit the
inflammatory-mediated increase in ASIC expression in sensory neurons
[114]. The proposed mechanism of action of the
NSAIDs is allosteric inhibition of channels by slowing recovery after
inactivation [140].

Aminoglycosides (streptomycin, neomycin, and gentamicin)
represent a group of antibiotics that have been shown to have a
blocking effect on a wide range of receptors: Ca2+ channels,
excitatory amino acid receptors, TRPV1 (transient-receptor-potential
V1) channels [141]. In DRG neurons, streptomycin
and neomycin at 30 µM concentration showed significant reversible
reduction in amplitude of ASIC currents and retarding action on the
desensitization process. In this case, decrease in concentration of
Ca2+ in the extracellular medium enhanced the effect of
streptomycin and neomycin on desensitization [115].

Diarylamidines are widely used initially to treat diseases caused
by protozoa such as trypanosomiases and leishmaniases. Four
representatives of the diarylamidines –
4,6-diamidine-2-phenylindole, diminazene, hydroxystilbamidine, and
pentamidine – inhibit currents generated by ASIC receptors in
hippocampal neurons with IC50 values of 2.8, 0.3, 1.5, and
38 µM, respectively. Diminazene also showed increased
desensitization of ASIC currents in hippocampal neurons. The inhibitory
effect of diminazene on ASIC currents in CHO cells decreases in the
series ASIC1b > ASIC3 > ASIC2a ≥ ASIC1a [117].

2-Guanidine-4-methylquinazoline (GMQ), a small molecule
containing a guanidine group and a heterocycle, activates ASIC3 at
neutral pH without any effects on ASIC1a, 1b, and 2a (EC50
value measured in CHO cells is 1 mM). In this case, the activated
channels have less pronounced selectivity for Na+. It was
proposed that conformational changes of transmembrane domains caused by
GMQ–channel interaction differ from conformational changes
induced by protons only. Using site-directed mutagenesis of the
channel, it has been shown that activation of the ASIC3 channel by the
GMQ molecule involve residues Glu79, Glu423, Leu77, and Arg376 located
in a small cavity that is located at the base of the extracellular palm
domain [118, 142]. GMQ
activates sensory neurons and causes pain associated with the
activation of ASIC3 [118].

Sevanol (or 9,10-diisocitryl ester of epiphyllic acid), isolated
from acetic extract of the Thymus armeniacus plant, is the first
natural low molecular weight compound that inhibits both components of
the current of the ASIC3 channel. The peak current component is
completely inhibited (IC50 353 µM), whereas the
inhibition of the sustained component is only 45% (IC50 of
234 ± 53 µM). At the same time, it also inhibits the
conductivity of ASIC1a expressed in frog oocytes, although less
efficiently. On models of the acid-induced pain and thermal
hypersensitivity caused by inflammation, sevanol at doses of
1-10 mg/kg exhibited a pronounced analgesic effect [120].

Polypeptide modulators. Natural venoms have long established
themselves on equal terms with plant extracts as indispensable sources
of biologically active compounds [143]. A series
of polypeptides that are capable of modulating the activity of ASICs
have been found in animal venoms. All currently known polypeptides are
markedly different both in primary and in the spatial structure. Four
spatial structures of polypeptide modulators from natural venoms with
different folding of polypeptide chains are shown in Fig. 4.

The greatest progress has been achieved in the study of
psalmotoxin1 isolated from the spider
Psalmopoeus cambridgei, which is a highly selective inhibitor of
the receptor of ASIC1a [125]. PcTx1 is a small
polypeptide consisting of 40 a.a., has a molecular weight of 4689.4 Da,
and has pronounced basic properties (pI 10.38, nine positively
charged amino acid residues). The spatial structure of PcTx1 is a
compact central core stabilized by three disulfide bonds, from which
N- and C-terminal loops protrude (Fig. 4). Such folding is named “inhibitory cysteine
knot” [144, 145].
Most of the toxins from spider venoms acting on ionotropic receptors
have this fold [146].

Natural PcTx1, as well as its synthetic analogs, have equally
high-affinity inhibitory effect on homomeric ASIC1a channels in various
cellular expression systems (IC50 < 1 nM). Binding
occurs at pH 7.4 with the channel existing in the closed state.
This process is reversible. Full blocking of the current is observed at
10 nM peptide concentration [125]. An interesting
fact is that at pH values above 7.4, application of PcTx1 can lead to
the activation of ASIC1a [147]. It should also be
noted that PcTx1 has a potentiating effect on ASIC1b [126].

Functional study of the toxin showed that Trp24, Arg26, and Arg27 play
significant roles in the activity of PcTx1 [148].
Studies of chimeric ASIC channels constructed from sensitive and
insensitive to PcTx1 subunits, computer modeling, and site-directed
mutagenesis of ASIC1a showed that the polypeptide is attached to the
channel in the acidic pocket that is responsible for pH-dependent
channel opening [148-150].
The crystal structure of the PcTx1 complex with the ASIC1a channel
demonstrated that both the hydrophobic region of the toxin (which
interacts with the thumb domain of the channel) and a positively
charged cluster (especially Arg26 and Arg27 which form hydrogen bonds
with residues in the acidic pocket) participate in the binding [151].

The mechanism of PcTx1 action involves increasing of ASIC1a affinity to
H+, and thus the toxin causes the desensitization of the
channel, which ultimately leads to the inability of the channel to be
activated [147]. This structural-functional study
of the highly specific ligand of ASIC1a defined the important role of
the acidic pocket in the pH-dependent opening of the channel.

The polypeptide MitTx was found in the venom of the coral snake
Micrurus tener tener. MitTx is an agonist of all functional
subtypes of ASICs, activating them at neutral pH. MitTx showed
different specificity towards different types of channels expressed in
Xenopus oocytes, in the nanomolar concentration acting on ASIC1a
and ASIC1b subtypes (EC50 = 9.4 ± 1.3 and 23
± 3.6 nM, respectively) and at micromolar concentrations
acting on ASIC2a and ASIC3 [124]. MitTx consists
of two components, MitTx-α and MitTx-β, which are
noncovalently associated with each other. MitTx-α is a 6-kDa
protein that is structurally similar to Kunitz-type protease
inhibitors, and MitTx-β is structurally similar to phospholipase
A2 [124]. The entire MitTx complex is
structurally similar to a toxin from a snake venom, β-bungarotoxin
(PDB 1BUN), which in contrast to MitTx inhibits K+
channels.

The crystal structure of the MitTx complex with ASIC1a channel revealed
that the toxin is attached to domains wrist, palm, and thumb, like a
“churchkey” bottle opener, wherein the binding region of
MitTx-β overlaps with that for PcTx1. Residues Phe14 and Lys16
play an important role in binding of MitTx-α. Phe14 interacts
with Ala82 and Thr84 in the β1-β2-linker of one subunit and
Val361 and Met364 of the other subunit. Lys16 interacts with a site on
the interface of the wrist and TM1 domains [152].

Conformational changes leading to the channel opening after toxin
binding occur in the bottom part of the palm domain, followed by
expansion of the extracellular pore region and rearrangement in the
α-helixes of the TM domains. At the same time, the region of
selective filter formed by Gly443, Ala445, and Ser446 extends parallel
to the membrane, breaking the helix of the TM2 domain into two parts,
and the carbonyl groups of Gly443 form a ring of radius ~3.6 Å
providing selectivity for the passage of Na+ ions [152].

Two more polypeptides, mambalgin-1 and mambalgin-2 (Ma-1
and Ma-2), were isolated from the venom of the snake Dendroaspispolylepis polylepis. They both consist of 57 a.a. and include
eight cysteines forming four disulfide bonds. The peptides differ by
one substitution in position 4, Tyr in Ma-1 or Phe in Ma-2. The
mambalgin structure includes tight central area stabilized by four
disulfide bonds (Cys3–Cys19, Cys12–Cys37,
Cys41–Cys49, and Cys50–Cys55) and three extended loops
forming two β-layers (Fig. 4) [153]. Such folding is named “three-finger
toxins”, typical for a large number of well-known neurotoxins
from snakes.

Mambalgins reversibly inhibit recombinant homomeric ASIC1a, heteromeric
ASIC1a/2a and ASIC1a/2b, as well as ASIC1b and ASIC1a/1b channels with
IC50 values of 55, 246, 61, 192, and 72 nM,
respectively [128]. It was also shown that they
inhibit the currents of ASIC channels in neurons of the spinal cord and
hippocampus and in sensory neurons. Mambalgins exhibited analgesic
effects in vivo in models of acute and inflammatory pain via
inhibition of ASIC1b channels in peripheral nervous system or via
inhibition of ASIC1a and ASIC1a/2a channels in CNS [128].

Mambalgins have a strong positive electrostatic potential on their
surface, which can play an important role in the binding of the
peptides to ASIC channels as in case of PcTx1. However, mambalgins have
a different mechanism of action, i.e. they bind to the channel in the
closed or inactivated state and change the channel affinity for protons
[128]. Functional studies of ASIC1a mutant
channel revealed the important role of residue Phe350 for the
interaction with mambalgins. It should be noted that this region
disposed in the vicinity from acidic pocket is considered important
also for the interaction with PcTx1. Thus, the binding sites for
mambalgins and PcTx1 overlap [154].

Today three polypeptides from different species of sea anemone with
inhibitory activity on ASIC3 are known. Two are referred to the most
numerous structural class 1, and the other to the rarest structural
class 9 [155].

Polypeptide APETx2, the main component of the venom of the sea
anemone Anthopleuraelegantissima, contains 42 a.a., has
mass of 4561.1 Da, and has basic properties (pI 9.59). This
polypeptide reversibly inhibits ASIC3 channels expressed in oocytes or
mammalian cells (IC50 = 63 nM), suppressing only
the peak current component [127]. The spatial
structure of the toxin was determined by NMR spectroscopy; it includes
a compact central area with three disulfides (Cys4–Cys37,
Cys6–Cys30, Cys20–Cys38) and protruding basic loop
(residues 15-27) as well as N- and C-termini (Fig. 4) [156]. By spatial structure,
APETx2 belongs to the defensins (β-defensin-like peptides)
comprising human antimicrobial peptides as well as some of the toxins
from venoms of snakes, sea anemones, and the platypus. Calculated on
the basis of the peptide structure dipole moment allows to specify a
basic-aromatic cluster on the surface of the toxin molecule formed by
amino acid residues Phe15, Tyr16, Arg17, Arg31, and Phe33, which
probably plays an important role in the interaction of APETx2 with the
channel [156]. This suggestion was partially
confirmed by studies showing that the replacement of Arg17 and Phe15
reduced the activity APETx2 towards the ASIC3 channel in mice by 25-
and 100-fold, respectively [157, 158].

APETx2 showed a significant analgesic effect in vivo in a model
of acid-induced muscle pain and in a model of peripheral inflammatory
pain in rats [13, 159]. It
should be noted that the effect of APETx2 is not selective, and
therefore an inhibition of Na+-channels by the toxin also
contributes to analgesia. APETx2 exerts an inhibitory effect on
voltage-sensitive Na+-channels Nav1.8
(IC50 2.6 µM for rat DRG neurons) [160] and Nav1.2 (IC50
110 nM for human channels expressed in Xenopus oocytes) [161].

A close structural analog of APETx2 – polypeptide Hcr 1b-1
– was isolated from alcoholic extract of the sea anemone
Heteractis crispa. It has a molecular weight of 4537 Da and
consists of 41 a.a., including six cysteine residues forming three
disulfide bonds. The peptide reversibly inhibits the peak component of
the current of human ASIC3 channels with an IC50 value of
5.5 µM [129].

Strictly structurally different polypeptide Ugr 9-1 was obtained
from the venom of the sea anemone Urticina grebelnyi. Its
molecular weight is 3135 Da, and it is the shortest polypeptide capable
of modulating the activity of ASIC. The spatial structure of Ugr 9-1 is
a β-hairpin and five β-turns stabilized by two disulfide
bonds, while a long N-terminus and a short C-terminus
protrude from the central area (Fig. 4) [130].

Ugr 9-1 has an inhibiting effect on the peak and sustained current
components of ASIC3 without affecting other types of acid-sensitive
channels. This distinguishes the biological properties of Ugr 9-1 from
other sea anemone toxins – APETx2 and Hcr 1b-1, which inhibit
only the peak current component. The peak component is completely
inhibited by addition of toxin (IC50 10 µM), and
the sustained component only by 48% (IC50
1.44 µM). In vivo Ugr 9-1 at doses of
0.1-0.5 mg/kg showed a significant analgesic effect (against
acid-induced pain and thermal hypersensitivity) [130].

CONCLUSION

ASIC channels are a group of proteins with extremely important
regulatory and sensory function for neurons of the peripheral and
central nervous system. Examples demonstrating involvement of these
channels (in particular ASIC1a and ASIC3) in physiological and
pathological processes are increasingly found. By a combination of
biochemistry and molecular biology, as well as through structural and
functional studies, it has been possible to make some progress in
establishing mechanisms of these channels, especially through the study
of the interaction of these receptors with their ligands. These studies
not only reveal the fundamentals of the functioning of these channels
but also are a strong foundation for the creation of effective drugs
for the treatment of a variety of pathological conditions.

This work was supported by the Russian Foundation for Basic Research
(grant Nos. 14-04-31578 and 12-04-01068), programs of the Presidium of
the Russian Academy of Sciences “Molecular and Cell
Biology” and “Fundamental Science for Medicine”, and
the Russian President Grant for State Support of Leading Scientific
Schools of the Russian Federation (NSh-1924.2014.4).